Obesity and its related morbities of type 2 diabetes mellitus and hypertension have reached epidemic proportions in adults and children in the United States. Most human obesity is inherited as a polygenic trait and, once such genetically predisposed individuals become obese, there is less than a 10% chance of producing permanent weight loss using medical interventions. Critical neural circuits within the hypothalamus contain specialized neurons which respond to metabolic and hormonal signals from the periphery to monitor peripheral metabolic function and adipose stores. Leptin is the most important of these hormonal signals. It interacts with specific neurons in the hypothalamic actuate (ARC) and ventromedial (VMN) nuclei as a negative feedback system to inhibit intake and increase energy expenditure. However, obesity-prone individuals are resistant to the inhibitory effects of leptin either before or after they become obese. We have selectively bred rats which rapidly develop diet-induced obesity (DIO) when the caloric density of their diets is increased. As with humans, DIO in these rats is a polygenic trait. Also, they have an inborn resistance to the anorectic and thermogenic effects of leptin before they become obese. This proposal focuses on the observation that altering the postnatal environment of both DIO and diet-resistant (DR) rats can alter their leptin sensitivity and propensity to become obese when fed high fat diets. We will utilize these rats, as well as LepRbEGFP mice in which neurons expressing the leptin receptor (Lepr-b) have a fluorescent tag and ob/ob mice without leptin and db/db mice with defective leptin signaling, to test the hypotheses that early manipulation of leptin and/or insulin intake from the dam are critical regulators of leptin sensitivity and that altered regulation of trafficking of the leptin receptor to the cell membrane may be a critical determinant of the inborn leptin resistance of DIO rats. AIMS 1 and 2 will define the anatomical and physiological manifestations and mechanisms underlying the increased leptin signaling and obesity resistance associated with raising DIO neonates in large litters (LL) by gavaging LL DIO or normal litter (NL) DR pups with leptin or insulin. This should counteract the effect of lowered levels of these hormones in LL DIO and NL DR dam milk as a postulated mechanism for the obesity resistance and increased leptin sensitivity of LL rearing on DIO rats. Similar studies will be carried out in LepRbEGFP mice to monitor the effects of LL rearing vs. leptin or insulin gavage on the development of specific neurons expressing Lepr-b. Also, the requirement for leptin signaling in producing the LL effect will be assessed in mice that produce no leptin (ob/ob) and those with defective leptin signaling (db/db) raised in LL or NL. Measures of leptin-induced anorexia and thermogenesis and increases in hypothalamic leptin receptor transcription, binding and downstream signaling pathways, food intake and body weight gain on chow and HE diet, carcass composition, expression of various mRNA and protein species will be used to identify the physiological, biochemical and molecular sites at which these manipulations act to increase leptin signaling. Additional studies will assess the requirement for leptin and insulin for the development of leptin signaling and physiological sensitivity in primary cultured ARC and VMN neurons. AIM 3 will focus on OB-RGRP. This transcript is generated from the same locus as the leptin receptor. It decreases Lepr-b trafficking to the plasma membrane and is overexpressed in DIO ARC. We postulate that this is an important mechanism underlying the marked reduction in DIO ARC and VMN leptin binding associated with the DIO rat's inborn leptin resistance to the anorectic and thermogenic effects of leptin. A lentiviral vector expressing OB-RGRP shRNA will be used to reduce the overexpression of OB-RGRP in the ARC (and/or VMN) to test the hypothesis that this will normalize the trafficking of Lepr-b to the cell membrane and make them obesity resistant. Similar studies will be carried out in neuronal cultures. The overall goal of this proposal is to utilize in vivo and in vitro models to explore the novel idea that increasing leptin sensitivity is a realistic therapeutic goal for the prevention and treatment of obesity-prone humans, particularly humans.